Lipopeptide-small molecule polypeptide supramolecular composition as well as preparation method and application thereof

Abstract

The invention discloses a lipopeptide-small molecule polypeptide supramolecular composition as well as a preparation method and application thereof. The lipopeptide-small molecule polypeptide supramolecular composition comprises a small molecule polypeptide active matter, amino acid and lipopeptide, based on the total weight of the composition, the composition comprises 0.1-5% of micromolecule polypeptide active matter, 0.1-10% of amino acid and the balance of lipopeptide. The lipopeptide-small molecule polypeptide supramolecular composition prepared by the invention is small in particle size and good in dispersity, has hydrophilicity and lipophilicity, can be self-assembled to form a vesicle structure with a structure similar to that of a cell membrane, and can further carry other active components.

Description

Technical Field

[0001] The invention belongs to the technical field of cosmetics and medicines, and specifically relates to a lipopeptide-small molecule polypeptide supramolecular composition and a preparation method and application thereof. Background Art

[0002] Supramolecular chemistry is based on molecular assemblies and intermolecular forces. Different types of intermolecular interactions can be distinguished based on their strength, orientation, and dependence on distance and angle. Based on the principle of supramolecular self-assembly, people can use intermolecular forces as a tool to assemble components or modules with specific structures and functions into new supramolecular compositions in a certain way.

[0003] Based on the designability of supramolecular materials, the molecules are self-assembled into a regular, structurally stable, multifunctional composite material by utilizing interactions such as hydrogen bonds, electrostatic attraction, and van der Waals forces formed between molecules.

[0004] Supramolecular compositions can be used for precise delivery of active ingredients. After encapsulating the active ingredients, they are flexible and deformable. After spontaneous deformation, they enter the skin gaps, allowing the active ingredients to penetrate the skin, improving the solubility of poorly soluble active ingredients, promoting transdermal absorption and oral absorption of active ingredients, and significantly improving the bioavailability of active ingredients. They have low-cost, high-efficiency applications in the fields of cosmetics, functional foods, and medicines.

[0005] At present, there is no report on the use of supramolecular composites composed of biologically active lipopeptides and small molecule polypeptides, and there is no report on their application in water to form vesicles for inclusion of active substances. Summary of the invention

[0006] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0007] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0008] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a lipopeptide-small molecule polypeptide supramolecular composition.

[0009] In order to solve the above technical problems, the present invention provides the following technical solutions: a lipopeptide-small molecule polypeptide supramolecular composition, comprising a small molecule polypeptide active substance, amino acids and lipopeptides;

[0010] Based on the total weight of the composition, the small molecule polypeptide active substance accounts for 0.1% to 5%, the amino acid accounts for 0.1% to 10%, and the lipopeptide is supplemented to 100%.

[0011] As a preferred embodiment of the composition of the present invention, the lipopeptide includes one or more of subtilisin, finogenin, palmitoyl oligopeptide, di(laurylamideglutamine)lysine, iturin, bacillusmycin, and polymyxin.

[0012] As a preferred embodiment of the composition of the present invention, the small molecule polypeptide active substance includes one or more of blue copper peptide, acetyl octapeptide-3, and acetyl hexapeptide-8.

[0013] As a preferred embodiment of the composition of the present invention, the amino acids include one or more of arginine, lysine, histidine, arginine ethyl ester, lysine ethyl ester, and arginine monoglyceride.

[0014] As a preferred embodiment of the composition of the present invention, the lipopeptide and the amino acid are combined together through proton exchange, hydrogen bonding and π-π interaction to form a supramolecular structure.

[0015] As a preferred embodiment of the composition of the present invention, the composition spontaneously forms a vesicle structure of 10 to 100 nm in water.

[0016] Another object of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing a composition, comprising:

[0017] Under an inert gas atmosphere, the lipopeptide, small molecule polypeptide active substance, amino acid and deionized water are fully mixed, and then heated and stirred for reaction;

[0018] After cooling to room temperature, the mixture is subjected to high pressure homogenization treatment at a pressure of 400 to 800 bar for 2 to 8 times, and then freeze-dried under vacuum conditions to obtain the composition.

[0019] As a preferred embodiment of the method for preparing the composition of the present invention, the heating and stirring reaction comprises the following steps: the heating temperature is 25 to 60° C., and the stirring reaction time is 12 to 24 hours.

[0020] Another object of the present invention is to overcome the deficiencies in the prior art and provide a composition for use in cosmetics and drug delivery and sustained release.

[0021] Beneficial effects of the present invention:

[0022] (1) All components of the lipopeptide-small molecule polypeptide supramolecular composition provided by the present invention are derived from biological fermentation and are green, non-toxic and non-irritating.

[0023] (2) The lipopeptide-small molecule polypeptide supramolecular composition prepared by the present invention is mainly composed of various amino acids and peptides, which have a structure similar to that of skin cells and are easily absorbed by the skin.

[0024] (3) The lipopeptide-small molecule polypeptide supramolecular composition prepared by the present invention has a small particle size, good dispersibility, and is both hydrophilic and lipophilic. It can self-assemble to form a vesicle structure with a structure similar to that of a cell membrane, and can further carry other active components.

[0025] (4) The lipopeptide-small molecule polypeptide supramolecular composition prepared by the present invention has excellent transdermal permeability, which can not only protect the biological activity of the small molecule polypeptide active substance, but also deliver it to the effective site, so as to give full play to the biological efficacy of the small molecule polypeptide active substance; under the protection of the lipopeptide-small molecule polypeptide supramolecular structure, the active substance takes effect quickly and is easily absorbed by the skin; it has a unidirectional effect, promotes the penetration of therapeutic drugs through the skin, and does not affect the in vivo process of endogenous substances; it has no interaction with excipients and active substances, and can effectively increase the transdermal absorption of drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0027] Figure 1 This is the H NMR spectrum of sample 1 prepared in Example 1 of the present invention.

[0028] Figure 2 This is the DSC curve of sample 1 in Example 1 of the present invention.

[0029] Figure 3 This is the TEM image of sample 1 in Example 1 of the present invention.

[0030] Figure 4 This is a diagram of the transdermal test results in Example 2 of the present invention.

[0031] Figure 5 This is a diagram of the transdermal test results in Example 3 of the present invention.

[0032] Figure 6 This is a graph showing the results of the MMP-1 inhibitory ability assay in Example 4 of the present invention. DETAILED DESCRIPTION

[0033] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0034] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0035] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0036] The raw materials in the present invention are all common commercially available products.

[0037] Example 1

[0038] The supramolecular composition was prepared according to the raw material ratio in Table 1:

[0039] (1) Under an inert gas atmosphere, a certain amount of lipopeptide, small molecule polypeptide active substance, amino acid and deionized water were thoroughly mixed in a flask, slowly heated to 50°C, and stirred for 24 hours;

[0040] (2) After cooling to room temperature, the mixture was subjected to high-pressure homogenization at 500 bar for 5 times, and then freeze-dried under vacuum conditions for 24 h.

[0041] (3) DSC experiment: Weigh a certain amount of sample into a standard sealed aluminum pan, and use an empty aluminum pan without sample as a blank sample; then heat the sample from -40°C to 60°C at a rate of 2°C / min, and record the heat change;

[0042] The entire experiment was carried out under a constant nitrogen flow rate of 30 mL / min.

[0043] Table 1 Raw material ratio table (unit: g)

[0044] sample 1 2 3 4 5 6 7 8 Subtilisin 89 86 86 92 97 88 82 92 Acetyl Hexapeptide-8 3 6 3 0 3 3 4 2 Arginine 8 8 11 8 0 9 8 6 Phase change temperature / ℃ 10.2 - - - - 31.1 12 21

[0045] Note: “-” indicates that the phase transition temperature was not measured.

[0046] The H NMR spectrum of sample 1 composition can be found at Figure 1 From the H NMR spectrum of sample 1, it can be seen that the terminal methyl group on sodium subtilisin, the hydrogen protons on the long aliphatic chain and the α proton of the peptide bond all appear in the composition of sample 1; at the same time, the hydrogen proton at the a position on arginine shifts from 3.2ppm to 3.6ppm after binding with subtilisin, indicating the emergence of a supramolecular structure.

[0047] The DSC curves of the composition of sample 1, subtilisin and arginine are shown in Figure 2 From the DSC graph, it can be found that single lipopeptide subtilisin or arginine has no phase transition behavior in the temperature range of -40℃ to 60℃, while sample 1 has an obvious exothermic peak at 10.2℃, indicating that sample 1 is different from single lipopeptide subtilisin or arginine and is a new structure.

[0048] Combining NMR and DSC experiments, it can be proved that samples 2-5 are dissolved without forming a supramolecular structure, while samples 1, 6-8 have a supramolecular structure different from that of a single subtilisin or arginine.

[0049] TEM image of sample 1 is shown in Figure 3 The results confirmed that the supramolecular composition has good water dispersibility and can self-assemble to form a vesicle structure with a wall thickness of 2.7nm. It can further carry other active components; the particle size is less than 100nm and remains highly uniformly dispersed.

[0050] Example 2

[0051] Transdermal experiment 1:

[0052] Sample A Acetyl hexapeptide-8 aqueous solution (concentration 300ppm) Sample B 1% aqueous solution of sample No. 1 in Example 1

[0053] Experimental methods:

[0054] In the whole experiment, intact and undamaged pig skin of equal thickness was selected for transdermal experiment. The pig skin was cut into pieces of about 2.5 cm × 2.5 cm in size and placed between the receiving pool and the supply pool of the Franz diffusion cell, with the stratum corneum facing the supply pool.

[0055] The receiving pool contained PBS buffer (pH=7.4) containing 5% Tween 80; 2 mL of the sample diluted 4 times was added to the supply pool; the assembled Franz diffusion cell was placed in the transdermal diffusion instrument, and the experimental conditions were set as follows: temperature was 37°C, stirring speed was 400 rpm, and transdermal time was 12 h.

[0056] After the transdermal test, the pigskin was taken out of the diffusion cell, and the residual sample on the surface of the pigskin was wiped clean with absorbent cotton. Then, the tape stripping method was used to determine the content of Dendrobium polysaccharide in the stratum corneum. 21 layers of skin on the surface of the pigskin were glued with 3M tape, the first layer was discarded, and the remaining 20 layers were placed in a 15mL centrifuge tube. 2mL of isopropanol was first added to the tube, ultrasonicated for 30min, and centrifuged at 5000rpm for 30min. The supernatant was filtered with a 0.22μm organic filter membrane, and the content of acetyl hexapeptide-8 was determined, which was the content of acetyl hexapeptide-8 in the stratum corneum.

[0057] The remaining skin surface was heated at 60°C for 15 seconds, and the viable epidermis was carefully cut off with a scalpel, shredded and placed in a 15 mL centrifuge tube, 2 mL of isopropanol was added, ultrasonicated for 30 minutes, centrifuged at 5000 rpm for 30 minutes, and the supernatant was filtered with a 0.22 μm organic filter membrane and the content of acetyl hexapeptide-8 was determined, which was the content of acetyl hexapeptide-8 in the viable epidermis.

[0058] The remaining skin was cut into pieces and placed in a 15 mL centrifuge tube. 2 mL of isopropanol was added and ultrasonicated for 30 min. Then, the supernatant was filtered with a 0.22 μm organic filter membrane and the content of acetyl hexapeptide-8 was determined. This was the content of acetyl hexapeptide-8 in the dermis.

[0059] The content of acetyl hexapeptide-8 was determined by high performance liquid chromatography.

[0060] The results of the transdermal test were as follows Figure 4 As shown, it can be seen from the results that the transdermal effect of acetyl hexapeptide-8 in the supramolecular composition is four times that of its aqueous solution. The system effectively improves the transdermal absorption effect of acetyl hexapeptide-8.

[0061] Example 3

[0062] Transdermal experiment 2:

[0063] Sample A: Sample No. 1 in Example 1 was diluted with pure water to a mass content of 1%, and madecassoside was added and stirred until it was completely dissolved. The madecassoside content was 0.5%.

[0064] Sample B: Madecassoside aqueous solution (mass concentration 0.5%)

[0065] Experimental methods:

[0066] In the whole experiment, intact and undamaged pig skin of equal thickness was selected for transdermal experiment. The pig skin was cut into pieces of about 2.5 cm × 2.5 cm in size and placed between the receiving pool and the supply pool of the Franz diffusion cell, with the stratum corneum facing the supply pool.

[0067] The receiving pool contained PBS buffer (pH=7.4) containing 5% Tween 80; 2 mL of the sample diluted 4 times was added to the supply pool; the assembled Franz diffusion cell was placed in the transdermal diffusion instrument, and the experimental conditions were set as follows: temperature was 37°C, stirring speed was 400 rpm, and transdermal time was 12 h.

[0068] After the transdermal test, the pigskin was taken out of the diffusion cell, and the residual sample on the surface of the pigskin was wiped clean with absorbent cotton. Then, the tape stripping method was used to determine the content of Dendrobium polysaccharide in the stratum corneum. 21 layers of skin on the surface of the pigskin were taped with 3M tape, the first layer was discarded, and the remaining 20 layers were placed in a 15 mL centrifuge tube. 2 mL of isopropanol was first added to the tube, ultrasonicated for 30 minutes, and centrifuged at 5000 rpm for 30 minutes. The supernatant was filtered with a 0.22 μm organic filter membrane, and the content of madecassoside was determined, which was the content of madecassoside in the stratum corneum.

[0069] The remaining skin surface was heated at 60°C for 15s, and the viable epidermis was carefully removed with a scalpel. The resulting pieces were placed in a 15mL centrifuge tube. 2mL of isopropanol was added, and the mixture was ultrasonicated for 30min. The mixture was centrifuged at 5000rpm for 30min. The supernatant was filtered through a 0.22μm organic filter membrane and the madecassoside content was determined. This was the madecassoside content in the viable epidermis.

[0070] The remaining skin was cut into pieces and placed in a 15 mL centrifuge tube. 2 mL of isopropanol was added and ultrasonicated for 30 min. Then, the mixture was centrifuged at 5000 rpm for 30 min. The supernatant was filtered with a 0.22 μm organic filter membrane and the content of madecassoside was determined. This was the content of madecassoside determined in the dermis.

[0071] The content of madecassoside was determined by high performance liquid chromatography.

[0072] The results of the transdermal test were as follows Figure 5 From the results, it can be seen that the lipopeptide-small molecule polypeptide supramolecular composition can self-assemble in water to form a vesicle structure, which can promote the dissolution and transdermal absorption of water-soluble active ingredients; compared with aqueous solution, this system effectively improves the transdermal absorption effect of hydroxymadecassoside.

[0073] Example 4

[0074] MMP-1 inhibitory activity detection: (using Sigma kit);

[0075] Sample A: aqueous solution of acetyl hexapeptide-8 (the mass concentration of acetyl hexapeptide-8 is consistent with that in sample C, 300 ppm);

[0076] Sample B: Nicotinamide mononucleotide aqueous solution (consistent with the mass concentration of nicotinamide mononucleotide in sample C, 0.5% mass fraction);

[0077] Sample C: Sample No. 1 in Example 1 was diluted with pure water to a mass content of 1%, and nicotinamide mononucleotide was added and stirred until it was completely dissolved, and the mass content of nicotinamide mononucleotide was 0.5%.

[0078] Experimental procedures

[0079]

[0080] Data processing:

[0081] Slope = (fluorescence value 2 - fluorescence value 1) / (time 2 - time 1)

[0082] Relative inhibition rate % = (slope 酶对照组 - Slope 样品组 ) / slope 酶对照组 ×100

[0083] Time 1 and Time 2 represent two time points within 30 minutes.

[0084] Fluorescence values ​​1 and 2 represent the fluorescence values ​​at the corresponding time points.

[0085] The experimental results are as follows Figure 6 As shown, the lipopeptide-small molecule polypeptide supramolecular composition can self-assemble in water to form a vesicle structure, which is beneficial to the dissolution and penetration utilization of the active substance. The MMP-1 inhibitory activity detection of sample C is significantly better than that of sample AB. This structure can promote the improvement of the bioavailability of water-soluble active substances.

[0086] Example 5

[0087] Essence formula:

[0088]

[0089]

[0090] The essence was divided into room temperature samples, heat-resistant samples, cold-resistant samples and light samples. The room temperature samples were placed away from light, the heat-resistant samples were placed in a constant temperature oven at (50±1℃), the cold-resistant samples were placed in a -18℃ refrigerator, and the light samples were placed in an artificial intelligence climate box. After 3 months, the sample properties, appearance, color and smell were observed. There was no oil-water separation phenomenon. The results are shown in the following table.

[0091] Table 9 Formulation stability test

[0092]

[0093] The experimental results show that after the sample provided by the present invention is compounded with the matrix, no obvious discoloration and stratification phenomenon occurs after being placed at room temperature, high temperature, low temperature and light for 3 months, which can meet the actual application needs.

[0094] In order to solve the problem of low utilization rate of active ingredients, the present invention effectively combines lipopeptide-small molecule polypeptide-amino acid to form a supramolecular composite, which is green, non-toxic and non-irritating; has a small particle size, good dispersibility, and is both hydrophilic and lipophilic; can be assembled into a vesicle structure with a structure similar to that of a cell membrane, and can further carry other active components; has excellent transdermal permeability, can not only protect the biological activity of the small molecule polypeptide active ingredient, but also can deliver it to the effective site, giving full play to the biological efficacy of the small molecule polypeptide active ingredient; the active ingredient takes effect quickly and is easily absorbed by the skin; has a unidirectional effect, promotes the penetration of therapeutic drugs through the skin, and does not affect the in vivo process of endogenous substances; has no interaction with excipients and active ingredients, and can effectively increase the transdermal absorption of drugs.

[0095] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the present invention.

Claims

1. A lipopeptide-small molecule polypeptide supramolecular composition, characterized in that: include, Small molecule peptide actives, amino acids and lipopeptides; Based on the total weight of the composition, the small molecule polypeptide active substance accounts for 0.1% to 5%, the amino acid accounts for 0.1% to 10%, and the lipopeptide is supplemented to 100%.

2. The composition according to claim 1, characterized in that: The lipopeptide includes one or more of subtilisin, finogenin, palmitoyl oligopeptide, di(laurylamide glutamine) lysine, iturin, bacillus, and polymyxin.

3. The composition according to claim 1 or 2, characterized in that: The small molecule polypeptide active substance includes one or more of blue copper peptide, acetyl octapeptide-3, and acetyl hexapeptide-8.

4. The composition according to claim 3, characterized in that: The amino acids include one or more of arginine, lysine, histidine, arginine ethyl ester, lysine ethyl ester, and arginine monoglyceride.

5. The composition according to claim 4, characterized in that: The lipopeptide and amino acid are combined together through proton exchange, hydrogen bond and π-π interaction to form a supramolecular structure.

6. The composition according to claim 1 or 5, characterized in that: The composition spontaneously forms a vesicle structure of 10 to 100 nm in water.

7. A method for preparing a composition according to any one of claims 1 to 6, characterized in that: include, Under an inert gas atmosphere, the lipopeptide, small molecule polypeptide active substance, amino acid and deionized water are fully mixed, and then heated and stirred for reaction; After cooling to room temperature, the mixture is subjected to high-pressure homogenization at a pressure of 400 to 800 bar, and then freeze-dried under vacuum conditions to obtain the composition.

8. The preparation method according to claim 7, characterized in that: The homogenization treatment is performed 2 to 8 times.

9. The preparation method according to claim 8, characterized in that: The heating and stirring reaction, wherein the heating temperature is 25 to 60° C., and the stirring reaction time is 12 to 24 hours.

10. Use of the composition according to any one of claims 1 to 6 in cosmetics and drug delivery and sustained release.

Images